Hematopoiesis can be viewed as a patterning process in which the pluripotent stem cell proliferates and differentiates into the various distinct cellular lineages of the blood system. Hematopoietic development is controlled largely at the level of transcription. A central question in the study of hematopoiesis is how distinct blood cell fates are specified by combinations of transcription factors. The Ets-family transcription factor PU.1 is expressed specifically in the lymphoid (B cells, NK cells) and myeloid (macrophages, neutrophils) lineages of the immune system. Targeted mutation of the PU. 1 gene in mice reveals that it is essential for the development of B cells and macrophages, while development of T cells and neutrophils is aberrant or delayed. The activity of PU.1 on target genes is concentration dependent: macrophage development requires high concentrations of PU.1 while B cell development requires low concentrations. Genes encoding cytokine receptors are a major class of PU.1 target genes in developing hematopoietic progenitors. For example, PU.1 is essential for transcription of the Interleukin-7 receptor alpha gene (IL- 7Ralpha) in developing lymphoid cells, and for the macrophage colony-stimulating factor receptor gene (MCSFR, encoded by c-fms) in developing myeloid cells. Taken together, this evidence suggests that PU.1 plays a major role in cell fate decisions of stem cells to develop into the lymphoid or myeloid lineages. The focus of this grant proposal is to elucidate the function of PU. 1 in lympho-myeloid specification and in B cell differentiation. First, we will investigate whether graded levels of PU.1 regulate lymphoid versus myeloid cell fate decisions made by hematopoietic stem cells and/or multipotential progenitors. We will use both in vivo and in vitro approaches to address this problem. Second, we will utilize recently developed PU. 1-1-pro-B cell lines to investigate whether PU.1 is required for B cell differentiation in addition to being required for early events in B cell development.